the conversion of the carcinogen iv-hydroxy-2 ... · laboratory, the report of booth and boyland...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 7, Issue of April 10, pp. 1729-1740, 1969

Printed in U.S.A.

The Conversion of the Carcinogen IV-Hydroxy-2- fluorenylacetamide to oAmidophenols by Rat Liver in Vitro

AN INDUCIBLE ENZYMATIC REACTION*

(Received for publication, September 18, 1968)

H. R. GUTMANN AND R. R. ERICKSON

From the Laboratory for Cancer Research, Veterans Administration Hospital, and the Department of Biochemis- try, University of Minnesota, Minneapolis, Minnesota 55417

SUMMARY

The enzymatic conversion of the carcinogenic arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to the o-amidophenols, N-(1-hydroxy-2-fluorenyl)acetamide and N-(3-hydroxy-2-fluorenyl)acetamide, referred to as the isomerization of N-hydroxy-2-fluorenylacetamide, by cell fractions of rat liver has been studied. Cell fractions prepared from the livers of normal rats were unable to perform the reaction. Isomerization was stimulated by 3-methylcholanthrene and several other polycyclic aromatic hydrocarbons administered 24 hours preceding the preparation of the cell fractions. Since DL-ethionine inhibited the stimulation of the formation of the o-amidophenols by 3-methylcholanthrene and since the inhibition was counteracted by the concurrent administration of DL-methionine, the isomerization appeared to be an induced enzymatic reaction. The isomerization proceeded optimally at a pH near 7 in phosphate buffer, Tris buffer, or deionized water and, in contrast to the microsomal p-hydroxylation of N-2-fluorenylacetamide, did not require a NADPH-generating system. N-2-Fluorenylacetamide, the substrate for the NADPH-dependent formation of the p- amidophenols, N-(7-hydroxy-2-fluorenyl)acetamide and N- (5hydroxy-2-fluorenyl)acetamide, did not serve as a substrate for the isomerization. The isomerization was dependent on the synergistic action of two components. One of these was inducible and associated with the microsomal fraction of rat liver. The second component, localized in the soluble fraction of normal rat liver, was not dialyzable and, upon gel titration of the soluble fraction on polyacrylamide gel, was eluted with the macromolecules excluded from the gel. Resolution of the soluble fraction on DEAE-cellulose into five subfractions and assay of each fraction indicated that the highest activity was associated with a group of proteins eluted with dilute sodium phosphate

* This investigation was support,ed by United States Public Health Service Research Grant CA-02571 from the National Can- cer Institute.

buffer at pH 4.6. The formation of the reduced metabolites, N-2-fluorenylacetamide and 2-fluorenamine, by each fraction paralleled the formation of the o-amidofluorenols. These results and the further observation that the products of the isomerization and of the reduction were present, in all fractions, in a constant proportion suggested a common intermediate for both reactions. On the basis of these data, the isomerization has been tentatively formulated as a two-step reaction. In the first step N-hydroxy-2-fluorenylacetamide is dehydroxylated to a positively charged amidonium ion by an enzyme in the soluble fraction of rat liver. The second step in which hydroxyl ions add to the electrophilic carbon atoms 1 and 3 of the resonance forms of the amidonium ion yields the o-amidofluorenols. The induced microsomal enzyme is presumed to participate in this step of the reaction. Alternatively, hydride ions may be transferred to the positively charged nitrogen of the amidonium ion to give N-2- fluorenylacetamide and, via deacetylation, E-fluorenamine.

The first evidence that the mechanism for the formation of the o-amidophenols, N-(1-hydroxy-2-fluorenyl)acetamide and N- (3-hydroxy-2-fluorenyl)acetamide from the carcinogen, N-2- fluorenylacetamide differed from a microsomal NADPH-dependent p-hydroxylation of the arylamide came from tracer experiments in vivo by Miller and Miller (1) and by Miller, Cramer, and Miller (2). These workers showed that the simul- taneous administration of N-hydroxy-FAA,’ a carcinogenic

1 The abbreviation used is: FAA, N-2-fluorenylacetamide. The hydroxylated derivatives are abbreviated by indicating the posi- tion of the hydroxyl group in the fluorene system or on the nitrogen atom as follows: 1-hydroxy-FAA, N-(1-hydroxy-2-fluorenyl)- acetamide; 3-hvdroxv-FAA, N-(3-hvdroxv-24uorenvl)acetamide: 5-hydroxy:FAA, N(&hydroxy-2-fl~orenyl)acetamid”e;’7-hydroxy~ FAA, N-(7.hydroxy-2-fluorenyl)acetamide; N-hydroxy-FAA, N- hydroxy-2-fluorenylacetamide or N-2-fluorenylacetohydroxamic acid.

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1730 Isomerixation of N-Hydroxy-Z-Jluorenylacetamide to o-Amidophenols in Vitro Vol. 244, No. 7

metabolite of FAA (3), and of FAA-9-14C resulted in the urinary excretion of 1-hydroxy-FAA with a very much lower specific radioactivity than that of 5-hydroxy-FAA and of 7-hydroxy-FAA which are the products of the microsomal p-hydroxylation of FAA (4, 5). They concluded that N-hydroxy-FAA, rather than FAA, was the immediate precursor of I-hydroxy-FAA and that the o-amidophenol might be formed in tivo via a rearrangement in a manner analogous to the transformation of arylhydroxyl- amines to phenolic amines in acid (6). The metabolic reaction leading to l-hydroxy-FAA was subsequently studied in vitro by Booth and Boyland (7). These investigators reported an enzyme in the soluble fraction of rat and rabbit liver which converted N-hydroxy-FAA to 1-hydroxy-FAA, provided that NAD, NADH, or NADPH was available. Since other arylhydroxamic acids, such as N-hydroxyacetanilide, N-hydroxy-2- acetamidonaphthalene, and N-hydroxy-4-acetamidobiphenyl, likewise yielded o-amidophenols under these conditions (7), the authors expressed the view that the isomerization of arylhydroxamic acids to o-amidophenols by a soluble enzyme requiring the above cofactors for activity was a general metabolic reaction. Because the binding of the o-quinone imines, derived from l- hydroxy-FAA and 3-hydroxy-FAA, to proteins and the relation- ship of the binding of these compounds to the biological activity of FAA and of N-hydroxy-FAA were then under study in this laboratory, the report of Booth and Boyland (7) was of great interest to us. However, the data presented by Booth and Boy- land were merely qualitative and thus permitted no conclusion as to the extent of the reaction. Accordingly, methods for the isolation, purification, and estimation of l-hydroxy-FAA and of 3-hydroxy-FAA in incubation systems of liver cell fractions with N-hydroxy-FAA were developed and applied to a detailed examination of the reaction. Although we were unable to confirm the observations of Booth and Boyland, evidence was obtained that the reaction is inducible, in rat liver, by certain polycyclic aromatic hydrocarbons and involves two enzymatic components. The characterization of the isomerization of N- hydroxy-FAA as an inducible enzymatic reaction forms the basis of this report.

EXPERIMENTAL PROCEDURE

Preparation and Properties of Labeled and Unlabeled Com- pounds2-N-Hydroxy-FAA-9-14C with a specific radioactivity of 1.42 x lOlo dpm per mmole was obtained from Tracerlab, Inc., Waltham, Massachusetts. The radiopurity of the arylhydroxamic acid was shown by descending chromatography on Whatman No. 1 paper with the upper phase of a mixture of cyclohexane-t-butyl alcohol-glacial acetic acid-water (16: 1: 1: 1) as the solvent. A scan of the developed chromatogram3 showed one radioactive peak, RF = 0.55, coincident with the single, fluorescence-quenching spot seen on exposure of the chromatogram to ultraviolet light (2537 A). For use in the experiments the labeled compound was diluted with unlabeled N-hydroxy- FAA to a specific radioactivity of 8.2 x lo8 dpm per mmole. Two preparations of FAA-9-14C from Tracerlab were used in

2 Melting points were taken on a Fisher-Johns melting point apparatus and are uncorrected. Ultraviolet and infrared absorption spectra were recorded on a Beckman DK-2A recording spectrophotometer and a Beckman IR-4 spectrophotometer, respectively.

3 Radiochromatograms were scanned with a scanner model RSC-BA, Atomic Accessories, Inc., Valley Stream, New York.

these studies. The labeled compounds were purified before use by preparative thin layer chromatography as described previously (8) and their specific radioactivities, after purification, were 3.79 x log dpm per mmole and 7.18 x lo9 dpm per mmole, respectively. The purified compounds, when rechromato- graphed on Silica Gel GF,,, with chloroform-methanol (98:2) or on Whatman No. 3MM paper with the above solvent system, gave single radioactive peaks with RF values of 0.30 and 0.44, respectively.

I-Hydroxy-FAA, m.p. 210-212” XM,e,tH 278 (E, 22,700) rnp (9), 3-hydroxy-FAA, m.p. 247-249”, XMm”,$!= 273 (e, 19,000), 279 (E, 18,500), 317 (E, 20,000) rnp (lo), 5-hydroxy-FAA, m.p. 217- 218”, XeazH 282 (E, 27,500), 298 (E, 23,700), 309 (E, 20,900) rnp (ll), 7-hydroxy-FAA, m.p. 230-232’, XMm”,$= 291 (e, 32,100) rnp (12), FAA, m.p. 196-198”, XE,zH 287 (E, 26,800) rnp (13) and 2-fluorenamine, m.p. 127-129”, XM,:zH 286 (E, 23,000) rnp (14) were prepared by published methods.

N-Acetoxy-2-jkorenylacetamide-A solution of N-hydroxy-FAA (15) (0.50 g, 2.1 mmoles) in freshly distilled acetic anhydride (10 ml) and pyridine (0.2 ml) stood at room temperature for 12 hours. The reaction mixture was then poured onto crushed ice. The N-acetoxy-FAA (0.48 g, 81% yield), m.p. 112-114”, which precipitated, was collected, washed with distilled water, and dried in a vacuum over phosphoric anhydride.

CnILsNO~ Calculated: C 72.58, H 5.37, N 4.98 Found: C 72.93, H 5.31, N 4.78

$,I: 1790 (0-C=O), 1690 (N-C=O) cm-i; XgitH 275 (E, 23,900), 289 (E, lS,SOO), 302 (E, 16,500) rnp.

Inducers of Hepatic Drug-metabolizing Enzymes and Co- factors-Benzo[a]pyrene, m.p. 175.5-177”, dibenz[a ,h]anthracene, m.p. 269-271”, testosterone, m.p. 153-156”, and 19nortestos- terone, m.p. 118-120”, were obtained from Sigma. 2-Chloro- lo-(3-dimethylaminopropyl)phenothiazine (SKF-2601), m.p. 54- 55”, and 4-chloro-19nortestosterone (SKF-6611), m.p. 176- 178.5”, were gifts of the Smith, Kline and French Laboratories. 5-Ethyl-5-phenylbarbituric acid, m.p. 177.5-178.5”, and the sodium salt of 5-ethyl-5-(1-methylbutyl)barbituric acid were purchased from Merck and Abbott Laboratories, respectively. The above compounds were judged to be pure on the basis of their melting points and they were used without further purification. 3-Methylcholanthrene m.p. 181-182”, and phenothiazine were purchased from Eastman Kodak, and the phenothiazine was recrystallized from butan-l-01 before use, m.p. 186-187”. The monosodium salt of NADP and the dipotassium or the disodium salt of glucose-6-P were products of Sigma. Nicotina- mide was purchased from Eastman Kodak.

Animals-Male albino rats weighing 160 to 220 g, obtained from the Holtzman Company, Madison, Wisconsin, were used in all experiments. The animals were given free access to com- mercial food pellets (Purina chow) and water except where noted otherwise in Table II.

Preparation of Cell Fractions-The animals were exsanguinated by decapitation and the livers were removed without prior

4 Lotlikar et al. (la), who first prepared N-acetoxy-2-fluorenylacetamide, reported a melting point of 109-111” for this compound. The compound was obtained by a somewhat different method of 0-acetylation of Nhydroxy-FAA and no elemental analysis was given.


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Issue of April 10, 1969 H. R. Gutmann and R. R. Erickson 173 I

20% homogenate of rat liver in 0.01 M phosphate buffer, pH 7.4, containing 1.1% KCI, centrifuged at 600 X g for 15 min

L Sediment

(discarded) 600 X g supernatant Tiyuid (Fraction A )

centrifuged at 105,000 X g for 60 min I

A Pellet (microsomes + mitochondria) washed

with 0.01 M phosphate buffer, pH 7.4, containing 1.1% KC1 and recentrifuged

at 105,000 X g for 30 min

I

A Soluble fraction

(Fraction C)

1 1 Microsomes + mitochondria Supernatant liquid

(Fraction B) (discarded)

FIG. 1. The preparation of the cell fractions of rat liver used in the assay of the isomerization of N-hydroxy-FAA

perfusion” and chilled on ice. All subsequent operations were carried out at 4”. The livers were washed with homogenizing buffer, blotted with filter paper, and minced with scissors. To equalize differences in enzyme content, the cell fractions for each experiment were obtained from the pooled livers of four to five rats. The mince was homogenized in a Potter-Elvehjem glass homogenizer equipped with a Teflon pestle (0.004-inch clearance). The homogenizing medium (4.0 ml per g of wet liver) was that described previously (4) except that the EDTA was omitted. In the experiments depicted in Fig. 6, a 1.1% solution of potassium chloride was used as the homogenizing medium. The resulting 20% homogenate was centrifuged at 600 X g for 15 min (Fig. 1) and the supernatant liquid equivalent to 4.0 g of wet liver (20 ml) was utilized in the incubations as the source of the isomerizing enzyme. In the experiments dealing with the intracellular localization of the enzyme 20 ml of the 600 x g supernatant liquid were fractionated, by centrifugation at 105,000 x g for 1 hour, into the microsomal-mitochondrial sediment and into the soluble fraction (Fig. 1). Prior to incubation the particulate fraction was dispersed in homogenizing medium, recentrifuged, and resuspended in 20 ml of homogenizing medium. In the initial experiments the incubation medium consisted of 15 pmoles of glucose-6-P, 360 pmoles of nicotinamide, 0.3 pmole of NADP, 150 pmoles of potassium chloride, and 150 pmoles of sodium phosphate buffer, pH 7.4 (Medium A). The

5 The livers were not perfused prior to removal since serum, when added to the microsomal-mitochondrial sediment from the livers of 3-methylcholanthrene-treated rats to give a serum protein concentration of 0.61 mg per ml of incubation system, converted 1.3% of the substrate, N-hydroxy-FAA-9-r4C, to l-hydroxy- FAA-g-r%. The soluble proteins from the same rat (5.7 mg per ml of incubation system), when tested separately and concurrently with the same cell fraction, yielded 15.4y0 of l-hydroxy- FAA-9.‘4C. These data indicated that the liver cell was the major source of the isomerizing activity. Note Added in Proof--This conclusion was reinforced by anot.her control experiment in which whole blood was homogenized, fractionated (Fig. l), and tested for the isomerization of Nhydroxy-FAA in the same way as the cell fractions from liver. Under these conditions the soluble proteins of liver (5.7 mg of protein per ml of incubation system) isomerized A’-hydroxy-FAAW4C to a 17.fold greater extent than did the processed whole blood (0.57 mg of protein per ml of incubation system).

microsomal hydroxylation of the fluorene nucleus proceeded optimally in this medium (4). In later experiments the compounds and cofactors necessary for hydroxylation of the aromatic ring were omitted from Medium A and the incubation medium consisted only of 40 pmoles of sodium phosphate buffer, pH 7.4 (Medium B). The appropriate cell fraction (2.0 ml, equivalent to 0.4 g of wet liver) and the substrate dissolved in 0.1 ml of methyl Cellosolve were added to Medium A or B to give a total volume of 6.1 ml per flask. A total of 10 flasks (containing the equivalent of 4.0 g of wet liver) was used routinely in each experiment. The flasks which were open to air were shaken in a water bath maintained at 37”. After 1 hour the contents of the flasks were combined and the proteins were precipitated by the addition of 6 volumes of ethanol. After the mixture stood at 4” for 0.5 hour, the precipitate was separated by centrifugaton and the metabolites in the supernatant liquid were partitioneid by solvent extraction as shown in Fig. 2. In the radioactive tracer experiments, the carrier compounds dissolved in methanol were added, with vigorous shaking, prior to the precipitation of the proteins.

Purification and Determination of Metabolites-The residue which remained after evaporation of the solvent of Fraction F-l (Fig. 2) was dissolved in methanol, and the solution was applied as a band (1 x 15 cm) to Whatman No. 3MM paper. Authentic I-hydroxy-FAA, 3-hydroxy-FAA, 5-hydroxy-FAA, 7-hydroxy- FAA, and N-hydroxy-FAA were spotted as markers at the origin. The chromatogram was developed with the upper phase of the solvent system described above after equilibration with both phases for 18 to 20 hours. The separated compounds (Fig. 3) were located by exposure of the chromatogram to ultraviolet light (2537 A), and the compounds were eluted by descending chromatography with methanol. I-Hydroxy-FAA and 3-hydroxy-FAA were further purified by thin layer chromatography on Silica Gel GF2s4 (20 x 20 cm plates, 0.25 mm thickness of adsorbent). Since 5-hydroxy-FAA and 7-hydroxy-FAA mi- grated on Whatman No. 3MM paper with identical rates, these compounds were separated subsequently by thin layer chromatography on plates (20 x 20 cm) coated to a thickness of 0.25 mm with silicic acid (Mallinckrodt, 200 mesh) mixed with calcium sulfate (13%) and zinc silicate (1%). The thin layer chromatograms were developed without previous equilibration


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1732 Isomerixation of N-Hydroxy-2;fluorenylacetamide to o-Amidophenols in Vitro Vol. 244, No. 7

Incubation system; deproteinized with 6 volumes of ethanol; centrifuged at 0” for 10 min

Precipitate (discarded)

Supernatant liquid, ethanol distilled at reduced pressure; volume adjusted to 60 ml; pH adjusted to 4; extracted

three times with 60 ml of ether

Ether extracted two times with 25 ml of 0.25 N NaOH; washed with water

Aqueous phase (discarded)

Ether (F-2) contains FAA and 2-fluorenamine; compounds separated by thin layer

chromatography

Aqueous phase washed with ether; acidified (pH 2) with 8 N HCl; extracted two

times with 25 ml of ether I

J. Ether (F-l) contains acidic-phenolic metabolites; washed with distilled water; solvent removed at

reduced pressure; residual compounds separated by paper chromatography

and by thin layer chromatography

&

Aqueous phase (discarded)

FIG. 2. The partition of the metabolites of N-hydroxy-FAA or of FAA by solvent extraction after incubation of these substrates with cell fractions from rat liver.

Origin

I, ’ I

t 1

I I f,

N-hydroxy -FAA

I- hydroxy -FAA

3-hydroxy-FAA

l 5-hydroxy-FAA+ 7- hydroxy-FAA

I FIG. 3. The separation of the acidic and phenolic compounds

in Fraction F-l (Fig. 2) by descending paper chromatography on Whatman No. 3MM paper with the upper phase of a mixture of cyclohexane-t-butyl alcohol-glacial acetic acid-water (16: 1: 1: 1) as a solvent. The bands were visualized by the quenching of the fluorescence on exposure of the developed chromatogram to ultraviolet light (2537 A) or by spraying with dilute (1:3) Folin reagent.

in glass tanks lined with Whatman No. 3MM paper. The mobilities of the compounds in various systems are listed in Table I. The compounds were eluted from the gel by repeated extractions with methanol (2 x 2.0 ml) and the ultraviolet

absorption spectra of the eluted compounds were checked against those of authentic samples. Purification by thin layer chromatography was repeated until the spectra of the isolated metabolites were superimposable on those of authentic samples. Th: quantities of 1-hydroxy-FAA and of 3-hydroxy-FAA in the extract of the final chromatogram were then determined by means of the appropriate extinction coefficients listed above. These values were corrected for loss of compound during the isolation and purification by means of experimentally determined values for the recoveries of the compounds. The recoveries were estimated by adding known amounts of the compounds to the complete incubation system and by precipitating the proteins immediately. The separation, purification, and estimation of the compounds were then carried out as described above. The average recoveries of 1-hydroxy-FAA and of 3-hydroxy-FAA in three different experiments were 27.1 f 1.6% and 32.6 f 2.8%, respectively. The lower limit for the spectrophotometric estimation of the isomerization of N-hydroxy-FAA to the o-

amidofluorenols was 0.30%. In the radioactive tracer experiments the carrier compounds, which had been isolated by solvent extraction and separated by paper chromatography, were purified to constant (j~lO%) specific radioactivity by repeated thin layer chromatography. The quantities of metabolite formed were calculated in the usual manner from the final

specific radioactivity and from the amounts of carrier added. Under the experimental conditions the isomerization of as little as 0.01% of N-hydroxy-FAAW4C was measurable by inverse isotopic dilution.

Resolution of Soluble Fraction of Rat Liver by Gel Filtration or Ion Exchange Chromatography and Assay of Resulting Fractions for Activity in Isomerization of N-Hyclroxy-FAA-Several


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Issue of April 10, 1969 H. R. Gutmann and R. R. Erickson

TABLE I

Mobilities of metabolites of N-hydroxy-FAA on thin layer chromatograms

1733

Compound tested

LHydroxy-FAA .................... 3-Hydroxy-FAA. ................... 7-Hydroxy-FAAc ............ FAA.............................:: 2-Fluorenamine ....................

-

Chloroform-methanol Ether-ethanol (9: 1) (9: 1)

0.59 f 0.08

0.55 f 0.06 0.54 f 0.05 0.66 f 0.01 0.70 f 0.07

0.69 f 0.05 0.66 f 0.03 0.58 f 0.06 0.62 f 0.04 0.65 f 0.05

0.42 zt 0.06 0.73 f 0.06 0.30 f 0.02 0.38 f 0.10 0.68 f 0.04 0.25 f 0.02 0.27 + 0.04d 0.60 f 0.06 0.11 f 0.03 0.35 f 0.03 0.66 f 0.03 0.36 f 0.08 0.55 + 0.03 0.75 f 0.04 0.56 f 0.07

* The values are the means and the average deviations from the means of five to 20 runs made with the authentic samples.

c This compound, although not a product of the isomerization of N-hydroxy-FAA, was used as a carrier in several of the experiments described in the text.

- Q The compounds were applied as spots on glass plates (20 x 20

cm) coated with 0.25 mm of Silica Gel GFzsa. The chromatograms were developed without previous equilibration by the ascending technique in glass tanks lined with Whatman No. 3MM paper. The compounds were located as fluorescence-quenching spots when the developed chromatograms were exposed to ultraviolet light (2537 A).

experiments were designed to characterize the soluble component in rat liver which participated in the isomerization of N-hydroxy- FAA as shown below. The soluble fraction obtained from 11.0 g of wet liver was subjected to gel filtration on Bio-Gel P-6.6 The gel was equilibrated overnight with 0.01 M phosphate buffer, pH 7.4, and was then poured into a column (3.3 x 44 cm). The soluble fraction was applied to the gel and elution was carried out with the above phosphate buffer at a flow rate of 90 ml per hour. Fractions of 9 ml were collected and the absorbances of each fraction at 260 and 280 rnp were determined with a Hitachi Perkin-Elmer model 319 spectrophotometer. The elution profile exhibited two peaks. The first of these which contained molecules with a molecular weight exceeding 4600 had an average value of 1.31 (range, 1.29 to 1.32) for the Atso:

AzeO ratio and was considered to contain primarily proteins. The effluent fractions comprising each peak were combined to give Fractions I and II, respectively. An aliquot (50 ml) of Fraction I (300 ml) was tested, in conjunction with the microsomal-mitochondrial sediment prepared from 4.0 g of wet liver of 3-methylcholanthrene-treated rats, for its ability to isomerize N-hydroxy-FAA. The metabolites, I-hydroxy-FAA and 3- hydroxy-FAA, were estimated spectrophotometrically. Frac- tion II was lyophilized and the residue was dissolved in 100 ml of 0.01 M sodium phosphate buffer, pH 7.4. The activity of an aliquot (50 ml) was assayed as described above for Fraction I.

In two experiments the soluble fraction was chromatographed on columns (3.5 x 50 cm) of DEAE-cellulose. The same technique had been used previously to isolate, from the soluble proteins of rat liver, a group of proteins associated with Shoulder B (fig. 4) which bind FAA-g-W (8) and N-hydroxy-FAA-9-14C7 preferentially under conditions in wivo. The soluble fraction prepared from 28.0 g of wet liver was dialyzed against 0.005 M

NaHC03, pH 9.5, until the pH within the dialysis bag was 9.5. The dialyzed solution (100 ml) was applied to the column and elution was carried out in a stepwise manner with 650 ml of each of the five buffers indicated in the legend of Fig. 4. The effluent,

6 This polyacrylamide gel was obtained from Bio-Rad. The exclusion limit of Bio-Gel P-6 is stated by the manufacturer to be 4600.

7 E. J. Barry, D. Malejka-Giganti, and H. R. Gutmann, unpub- lished experiments.

Rp values in a. *

Benzene-ethyl acetate (3:7)

Ether-methanol (9: 1)

d 7-Hydroxy-FAA and 5-hydroxy-FAA when chromatographed in this solvent system on silicic acid (Mallinckrodt, 200 mesh) had RF values of 0.40 and 0.49, respectively.

which emerged at a rate of 190 ml per hour, was collected in 16-ml fractions and the absorbances of each fraction at 260 and 28j0 rnp were determined. The elution profile exhibited four major peaks (A, C, D, and E, Fig. 4) and a shoulder, B. The resolving power of the fractionation was indicated by the concentration of the hemoproteins, measured by their absorbance at 420 rnp (Fig. 4), in Pedc C. The individual fractions, comprising each peak and the shoulder, were combined to give Fractions A, B, C D , and E, respectively. In agreement with previous data (8) the recovery of the proteins from the columns, as calculated from calorimetric measurements of the protein content of the five fractions by the modified Folin method (8, 17), was 48 %.

Radioactivity Measurements-The radioactivity of samples soluble in organic solvents was determined in a scintillator solution (10 ml) consisting of 2,5-diphenyloxazole (4 g) and 1,4-bis[2-(phenyloxazolyl)]benzene (0.1 g) in toluene (1 liter) The radioactivities of aqueous solutions were determined a. described previously (8). All samples were counted in duplicats with a liquid scintillation spectrometer at a level of confidence of at least 95%. Corrections for quenching were made by thee channel ratio procedure (18).

RESULTS

Incubation of N-hydroxy-FAA with the soluble fraction or with the 600 x g supernatant liquid of rat liver in Xedium A which supported the formation of 7-hydroxy-FAA by microsomes of rat liver (4) gave only trace amounts of I-hydroxy-FAA and of 3-hydroxy-FAA (Table II). The amounts of the o-amidofluorenols formed under these conditions were of the same order of magnitude as the quantities formed when N-hydroxy-FAA was incubated in 0.01 M phosphate buffer, pH 7.4, in the absence of any cell fraction. The detection of small amounts of l-hydroxy-FAA and of 3-hydroxy-FAA in incubation systems of

N-hydroxy-FAA and of cell fractions of rat liver was therefore referable to a nonenzymatic isomerization. The formation of sizeable amounts of 7-hydroxy-FAA observed on incubation of

the 600 x g supernatant liquid with N-hydroxy-FAA in Medium A was due to the microsomal hydroxylation of FAA which was present in the incubation systems and isolated from Fraction F-l (Fig. 2). The enzymatic reduction of N-hydroxy-FAA to FAA by rat liver homogenates (19) and the microsomal, NADPH-


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1734 Isomerixation of N-Hydrozy-d-#uorenylacetamide to o-Amidophenols in Vitro Vol. 244, No. 7

A420 o -..____... 0

2 e

2 2.0 - 5

0 4b ' sb Ii0 . ' 260 ' 240 Tuba Numbsr

1.2 6.0

3 c 0 5 1.0 q I-hydroxy-FAA+3-hydroxy-FAA .?I

P 3 E 3

0.8 4.0

< = .

2-k

g 3

$b 0.6 Sl

e E ""E

'OL 'b ST P 0.4 2.0 s+

G 2 0 4 0.2

E F

i 0 l-I I-I -I I- .I I-I 0

A B c

FIG. 4. DEAE-cellulose chromatography of the soluble fraction of rat liver (upper) and the relative magnitudes of the isomerixation and of t.he reduction of N-hydroxy-FAA by Subfractions A, B, C, D, and E combined with the microsomal-mitochondrial sediment from the livers of 3-methylcholanthrene-treated rats (lower). The soluble fraction from 64 g of wet liver was obtained as described in Fig. 1 and dialyzed against 0.005 M NaHC03 buffer, pH 9.5, for 20 hours prior to ion exchange chromatography. Pro- tein elution was carried out in a stepwise manner with 650 ml of each of five buffers. The numbers on the top of the figure correspond to the following buffers: I, 0.005 M NaHCOs, pH 9.5; ZZ, 0.02 M Na*HI’Od, pH 9.2; ZZZ, 0.02 M NaQHPOk, pH 8.5; IV, 0.07 M

NaHzPOd, pH 4.6; V, 0.05M NaHzPOh + 1.0~ NaCl, pH 4.6. Frac- tions of 1G ml per tube were collected at a rate of 200 ml of effluent per hour and the absorbances of each fraction at 260 and 280 rnp

D E ’

were determined. The contents of Tubes 16 to 40 (Subfraction A, A280:A280 = 1.68), Tubes 66 to 85 (Subfraction B, Aes,,:Aneo = 1.45), Tubes 86 to 120 (Subfraction C, A~go:A2~0 = 1.51), Tubes 124 to 184 (Subfraction D, Azs,,:Az~o = 1.73), and Tubes 185 to 214 (Sub- fraction E, Azso:A~so = 0.93) were dialyzed for 20 hours against 0.01 M sodium phosphate buffer, pH 7.4. Aliquots (55 ml) of each of the dialyzed subfractions were incubated with the microsomal- mitochondrial sediment from 4.0 g of wet liver of 3-methylcholanthrene-treated rats and with N-hydroxy-FAA-9-14C (0.14 rmole per ml of incubation mixture). The amounts of 1-hydroxy-FAA, 3-hydroxy-FAA, FAA, and 2-fluorenamine (FA) were determined by inverse isotopic dilution. In the figure I-hydroxy-FAA and 3.hydroxy-FAA are referred to as the o-hydroxylated metabolites and FAA and 2-fluorenamine as the reduced metabolites.

dependent hydroxylation of FAA to 7-hydroxy-FAA have already been described (4, 5). Attempts to stimulate the isomerization of N-hydroxy-FAA by the oral administration of

F,4A for 8 weeks at a level which induced the N-hydroxylation of FAA in viz10 (3) were unsuccessful. However, the yields of the o-amidofluoreuols in Medium A (Experiment 6, Table II) were increased about g-fold by a single, intraperitoneal injection

of 3methylcholanthrene to the rat 24 hours prior to the preparation of the 600 x g supernatant liquid. A 15- to 20.fold increase in the formation of I-hydroxy-FAA was observed when 2- diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A)8 in concentrations of lOA3 M or 5 x low3 M was added to the incubation systems which contained the 600 x g supernatant

8 We thank Dr. H. T. Nagasawa of this laboratory for supplying us wit,h this compolmd.

liquid from the livers of 3-methylcholanthrene-treated rats. Since SKF 525-A inhibits the NADPH-dependent, microsomal hydroxylation of aromatic compounds to some extent (20), the isomerization of N-hydroxy-FAA to 1-hydroxy-FAA appeared to be essentially different from the microsomal hydroxylation of FAA which takes place on carbon atom 7 of the fluorene nucleus and requires NADPH or a NADPH-generating system (4, 5). This view was strongly supported by the finding that the induced formation of the o-amidofluorenols proceeded optimally in media, such as phosphate buffer, Tris buffer, or deionized water, which lacked the compounds necessary for aromatic hydroxylations (Experiments 9, 10, and 11, Table II). It should also be noted that no 7-hydroxy-FAA was detectable in the incubation systems under these conditions (Experiment 9, Table II). The possibility that FAA, arising via the enzymatic reduction of N-


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TABLE II

1735

Eflect of conditions of incubation and of preliminary treatment of rat on extent of isomerization of 14C-labeled N-hydroxy-FAA to i-hydroxy-FAA and S-hydroxy-FAA by cell fractions of rat liver

The substrate concentration in all experiments was 0.070 wmole per ml. The tissue fractions were obtained from 4.0 g of wet liver. Except for the spectrophotometric estimation of 7-hydroxy-FAA in Experiment 8 the formation of the metabolites was determined by inverse isotopic dilution.

Experiment and cell fraction

1. None 2. 105,000 X supernatant liquid g 3. 600 X g supernatant liquid 4. 600 X g supernatant liquid 5. 600 X g supernatant liquid 6. 600 X g supernatant liquid 7. 000 X g supernatant liquid 8. 600 X g supernatant liquid

9. 600 X g supernatant liqaid 10. GO0 X g supernatant liquid 11. 600 X g supernatant liquid

a Not determined.

Preliminary xatment of rat

None None None 2.FAA” 3-MC” 3.MCc 3-MC”

3-MCc 3-MCc 3-MCc

.s

-

Incubation medium

Medium B Medium A Medium A Medium B Medium A Medium A

Medium A + 1OP M SKF 525-A Medium A + 5 X lo+ M SKF

525-A Medium B

Deionized water 0.01 M Tris buffer, pH 7.4

-

b FAA was administered in the diet at a level of 0.03°]0 (3) for 8 \veeks prior t.o the preparation of the cell fraction.

c 3-Methylcholanthrene (3-MC) (2.0 mg/lOO g of body weight) was injected intraperitoneally in 0.35 ml of corn oil 24 hollrs prior to the preparation of the cell fraction.

h\-drosy-FAA, served as the substrate in the isomerization of N-hydroxy-FAA was ruled out by esperiments in which N-hy- drosy-FA,2-9-14C and FAA-9-14C were incubated separately and concurrently, in Medium 13, with equal portions of the 600 x g supernatant liquid from the livers of 3-methylcholanthrene- treated rats. Scans of the paper chromatograms of Fraction F-l prepared from the incubation systems with N-hydroxy-FAA-g- 14C as the substrate showed a radioactive peak, RF = 0.29, coincident with authentic 1-hydroxy-FAA (Fig. 5). In contrast, no radioactive peak was detected in this region on chromatograms obtained from incubation systems which contained FAA-g-l% as the substrate. The possibility that N-acetoxy-2- fluorenylacetamide, a hypothetical intermediate in the metabo- lism of N-hydroxy-FAA and the precursor of a highly reactive amidonium ion (IS), was the substrate of the isomerization was also examined. In these experiments the formation of l-hydroxy-FAA and of 3-hydroxy-FAA by an aliquot of the 600 x g supernatant liquid from the livers of 3-methylcholanthrene- treated rats was compared with the formation of the o-amidofluorenols by another aliquot of the same cell fraction (Experi- ments 2 and 3, Table III). The data indicated that 2.7 and 0.5% of N-acetoxy-2-fluorenylacetamide were metabolized to l-hydroxy-FAA and 3-hydroxy-FAA, respectively. These values take account of the spontaneous conversion of N-acetoxy-2- fluorenylacetamide to l-hydroxy-FAA and 3-hydroxy-FAA which amounted to 4.6 and 4.4$& respectively (Experiment 1, Table III). Since N-hydroxy-FAA, under the same conditions and with the same cell fraction, yielded 7.8 and 5.1y0 of l-hydroxy-FAA and of 3-hydroxy-FAA, respectively, the arylhydroxamic acid was a better substrate for the induced formation of the o-amidofluorenols than was the acetate ester.

The isomerization of N-hydroxy-FAA was dependent on pH

Conversion of N-hydroxy-FAA

l-o I-hydroxy-FAA To 3.hydroxy-FAA / To ‘I-hydroxy-FAA

0.33 0.12 0.16 0.27 0.28

1.81 f 0.90” 5.57 f 0.27’ 4.87 f 0.17e

6.31 f 2.571 7.57 7.66

%

0.29 0.36 0.28 -Q 0.38

1.90 f 0.93” -a -Q

4.G8 f 1.2Gl 2.94 3.53

-c1

0.01 1.66 0.29 2.00

2.73 +c 0.74d -a -a

-0 -a -a

d This value is the mean and the average deviation from the mean of 11 experiments.

e This value is the mean and t)he average deviation from the mean of two experiments.

J This value is the mean and the average deviat.ion from the mean of nine experiments.

Q Not detectable by the spectrophotometric assay.

N-hydroxy -FAA fq=0.50

t I

24 -

22 -

‘,;-.,A 0 2 4 6 8 IO 12 14 16 18 20 22 24 26 28 35 37

Distance From Origin (cm)

FIG. 5. Evidence for the specificity of N-hydroxy-FAA as a substrate in the formation of 1-hydroxy-FAA by the 600 X g supernatant liquid. Equal amounts of the 600 X g supernatant liquid, prepared from the livers of 3-methylcholanthrene-treated rats as described in the text, were incubated in Medium B for 1 hour (a) with N-hydroxy-FAA-9-14C (4.2pmoles, 4.0 X 106 dpm) or (b) with FAA-S-l&C (4.2 rmoles, 4.2 X 106 dpm). The incubation mixtures were processed by solvent extraction as shown in Fig. 2. Frac- tion F-l was chromatographed on Whatman No. 3MM paper by the descending technique with the upper phase of cyclohexane-t- butyl alcohol-glacial acetic acid-water (16: 1: 1: l), and the chromatograms were scanned as described in the text. The upper tracing shows the distribution of the radioactivity when N-hydroxy-FAA-9.14C was the substrate. The lower tracing is a scan of the chromatogram when FAA-g-*%! was used as the substrate.


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1736 Isomerixation of N-Hydroxy-2-jhorenylacetamide to o-Amidophenols in Vitro Vol. 244, No. 7

TABLE III Relative yields of i-hydroxy-FL4A and of 3-hydroxy-FAA formed

from 1\T-acetoxy-2-$uorenylacetamide (N-acetoxy-FAA) and from Shydroxy-FAA by 600 X g supernatant liquid of

Z-methylcholanthrene-treated rats

Conversion of substrate

Experiment and substratea Incubation Preliminary

medium treatment of rats TO TO

l-h;dr;6xy- 3-h;pLbxy-

% 1. N-Acetoxy-FAA. B None 4.63 4.40 2. AT-Acetoxy-FAA. B 3-MC” 7.32 4.91 3. WHydroxy-FAA.. B 3-MC” 7.80 5.09

o The concentrations of M-acetoxy-FAA and of N-hydroxy- FAA in t.he incubation systems were 0.12 @mole per ml and 0.14 pmole per ml, respectively.

I, The qllantities of 1-hydroxy-FAA and of 3.hydroxy-FAAwere est,imated spectrophotometricnlly as described in the text.

c 3-Methylcholanthrene (3-MC) (2.0 mg/lOO g of body weight) was injected intraperitoneally in 0.35 ml of corn oil 24 hours prior to the preparation of the cell fractions.

II 0.80

f 1-hydmxy-FAA

pH of Incubation M&dlum

FIG. 6. Dependence of the isomerization of N-hydroxy-FAA to I-hydroxy-FAA and 3-hydroxy-FAA by the 600 X g supernatant liquid on pH. The 600 X g supernatant liquid was prepared from the livers of rats previously treated with Y-methylcholanthrene. The livers (20 g) were homogenized in deionized water (80 g) containing 1.1% KCl. Aliquots of the 600 X g supernatant liquid equivalent to 4.0 g of wet liver were incubated with A- hydroxy-FAA (0.14 rmole per ml of incubation mixture) in (a) 0.03 M acet,ate buffers (pH 4.0 and 5.0), (b) 0.03 M phosphate buffers (pH 6.0, 7.0, and 8.0), and (c) 0.03 M Tris buffer (pH 9.0). The determination of 1-hydroxy-FAA and of 3-hydroxy-FAA was carried out spectrophotometrically as described in the text.

(Fig. 6). The pH optimum, located near 7, and the shape of the curves relating pH and activity were identical for the conversion of N-hydroxy-FAA to I-hydroxy-FAA and 3-hydroxy-FAA. These data suggested that the same enzyme was involved in the formation of the two metabolites.

In addition to 3-methylcholanthrene, several compounds which have been described as inducers of microsomal drug- metabolizing enzymes in liver were tested, by single intraperitoneal injections, for their ability to induce the isomerization of N-hydroxy-FAA (Table IV). Benzo[a]pyrene and dibenz[a,h]- anthracene which induce microsomal hydroxylases acting on 2-amino-5-chlorobenzoxazole (zoxazolamine) (21) and on benz[a]pyrene (22), respectively, were at least as effective as 3-methylcholanthrene in inducing the isomerization of N-

hydroxy-FAA. Phenothiazine, which stimulates the hydroxylation of benz[a]pyrene (23), was less active than 3-methylcholanthrene, and the other compounds were inactive. Since the induction of hydroxylating activity by 5-ethyl-5-phenylbarbituric acid requires the repeated administration of the compound (al), the evidence for its inactivity in stimulating the isomerization of N-hydroxy-FAA is not entirely conclusive.

The isomerization of N-hydroxy-FAA was further characterized as an inducible reaction by the effect of nL-ethionine on the conversion of N-hydroxy-FAA to the o-amidofluorenols (Fig. 7). DL-Ethionine, when administered to the rat by intraperitoneal injection simultaneously with 3-methylcholanthrene 24 hours prior to the preparation of the 600 x g supernatant liquid, depressed the formation of I-hydroxy-FAA and of 3- hydroxy-FAA by 67 and 62%, respectively. The yields of the o-amidofluorenols were restored to 80% of the control values, when an amount of DL-methionine equal to that of m-ethionine was injected concurrently with the 3-methylcholanthrene and the nL-ethionine (Fig. 7). Similar effects by DL-ethionine, an inhibitor of hepatic protein synthesis (24), on the induction of demethylating and hydroxylating enzymes in the microsomes of rat liver have been reported (5, 22, 25). These inhibitions were likewise reversed by the administration of equimolar amounts of m-methionine (5, 22, 25).

The intracellular distribution of the isomerizing enzyme was examined by differential centrifugation of the 600 x g supernatant liquid and by assay of the mitochondrial-microsomal sediment from the livers of untreated or 3-methylcholanthrene-

TABLE IV Action of inducers of hepatic drug-metabolizing enzymes on isom-

erization of N-hydroxy-FAA to i-hydroxy-FAA and 3-hydroxy-FAA by 600 X g supernatant liquid from rat liver

The 600 X g supernatant liquid was obtained from 4.0 g of wet liver and was incubated with N-hydroxy-FAA (0.14 rmole per ml of incubation mixture) in Medium B. The quantities of l- hydroxy-FAA and of 3-hydroxy-FAA were determined spectrophotometrically as described in the text.

Experiment and inducer administered a

1. Benzo[a]pyrene.. 2. Dibenz [a, hlanthracene 3. Phenothiazine 4. 2.Chloro-IO-(3.dimethylamino-

propyl)phenothiazine (SKF- 2601).

5. 5-Ethyl-5-phenylbarbitaric acid 6. Sodium 5-ethyl-5(1-methyl-

butyl)barbitllrate 7. Testosterone. 8. 19-Nortestosterone. 9. 4-Chloro-19-nortestosterone

(SKF-GUI).

Amount of inducer

rdministereda

ng/lOO g, bad: weight

2.5 2.8 2.0

3.5 2.4

2.3 2.9 2.7

3.7


14.65 9.45 7.44 4.36 2.90 1.60

<0.30 <0.30

<0.30 <0.30 <0.30

<0.30

<0.30 <0.30

<0.30 <0.30 <0.30

<0.30

%

a Each compound was administered as a suspension in 0.35 ml of isotonic 0.9% NaCl containing 1.750/, acacia. The single injections were given 24 hours prior to the preparation of the homogenates.


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treated rats after recombination with the soluble fraction from the livers of untreated or 3-methylcholanthrene-treated rats (Table V). Neither the soluble fraction nor the microsomal- mitochondrial sediment from the livers of 3-methylcholanthrene- treated rats alone was able to isomerize N-hydroxy-FAA. How- ever, the microsomal-mitochondrial sediment from the livers of 3-methylcholanthrene-treated rats, in conjunction with the soluble fraction from the livers of untreated or 3-methylcholanthrene-treated rats, performed the reaction at a rate comparable to that of the 600 X g supernatant liquid of 3-methylcholanthrene-treated rats. It was evident that the isomerization of N-hydroxy-FAA required the synergistic action of two components. One of these was inducible by certain polycyclic aromatic hydrocarbons and resided in the particulate fraction of the liver cell, while the second component was noninducible and present in the soluble fraction of rat liver. Assays of mitochondria and of microsomes which had been separated by differential centrifugation at 10,000 X g (0.25 hour) and at 105,000 X g (1 hour) indicated that 33% of the isomerizing activity was associated with the mitochondria and 67a/, with the microsomes. This distribution of the activity suggested that the inducible component was a microsomal enzyme. Since the separation of the mitochondria from the microsomes by differential centrifugation is incomplete (26), the activity in the mitochondria was likely due to contamination with microsomes.

The noninducible soluble component of the isomerizing activity was investigated by dialysis experiments, by gel filtration, and by ion exchange chromatography of the soluble proteins. In these experiments the assay systems consisted of the dialyzed or fractionated macromolecules to which standard amounts of the microsomal-mitochondrial sediment from the livers of 3- methylcholanthrene-treated rats were added. Dialysis experiments indicated that virtually none of the isomerizing activity was dialyzable (Table VI). The soluble component was further characterized as a compound with a molecular weight >4600 by gel filtration of the soluble fraction on polyacrylamide gel. In these experiments 90% of the activity recovered in the effluent was found in the fraction excluded from the gel which contained the compounds with a molecular weight in excess of 4600.

Evidence that the soluble component of the isomerizing activity was associated with a protein or proteins was provided by the fractionation of the soluble proteins of rat liver on DEAE- cellulose which yielded the five subfractions (A, B, C, D, and E) previously described (8). Measurements of the Azso:/12~0 ratios confirmed that Subfractions A, B, C, and D which were eluted from the ion exchanger with buffers of increasing ionic strength and decreasing pH consisted of proteins (8). Subfraction E which was eluted with 1 .O M sodium chloride at pH 4.6 contained nucleic acids in addition to proteins. By far the largest amounts of I-hydroxy-FAA and 3-hydroxy-FAA were produced by the assay systems which contained the acidic proteins eluted at pH 4.6 (SubfracGon D). It may also be seen that the reduction of N-hydroxy-FAA to FAA and 2-fluorenamine proceeded optimally in this assay system and that the yields of these metabolites paralleled those of the o-amidofluorenols in each fraction (Fig. 4). Reduction of N-hydroxy-FAA to FAA by liver homogenates and cell fractions has been reported previously (19, 27). The 2- fluorenamine arose either by deacetylation of FAA (1) or by reduction of 2-fluorenylhydroxylamine (15). The sum of FAA and 2-fluorenamine represented therefore the total reduced metabolites derived from N-hydroxy-FAA. Subfraction D also

s I n I-hydroxy-FAA 1

q 3-hydroxy-FAA

3-MC f + +

M&thionino - - +

Ethioninc - + +

FIG. 7. The effect (a) of the intraperitoneal administration of on-ethionine (85 mg/lOO g of body weight) and (5) of the simulta- neous administration of on-ethionine (85 mg/lOO g of bodv weight) I. - ” YI and of nL-methionine (85 mg/lOO g of body weight) on the isomerization of N-hvdroxv-FAA to I-hvdroxv-FAA and 3-hvdroxv- FAA by the 600 X g supkrnatant liqui’h from the livers of 3-&$;I- cholanthrene-treated rats. 3-Methylcholanthrene (&WC) (2.0 mg/lOO g of body weight) was injected 15minafter the administration of the amino acids. The rats were killed 24 hours following the administration of the compounds and the 600 X g supernatant liquid was prepared from 4.0 g of wet liver as described in the text and in Fig. 1. The incubations were carried out in Medium B and the concentration of N-hydroxy-FAA was 0.070 rmole per ml

TABLE V

Evidence for participation of two components in isomerization of M-hyclroxy-FAA to 1 -hydroxy-FAA and 3-hydroxy-FAA by

rat liver

Experi- ment

1

2

3 4

5a

5b

Assay of cell fractions or combination of cell fractions’

Obtained from the Obtained from the 3-MC-treated ratb untreated rat

600 X g supernatan liquid

Microsomal-mitochondrial sediment

Soluble fraction Microsomal-mito-

chondrial sediment + soluble fraction

Soluble fraction


t


Soluble fraction


%

6.31 4.68

:0.30 <0.30

0.58 <0.30 5.20 3.56

:0.30

6.73

<0.30

3.87

a Each of the fractions was obtained from 4.0 g of wet liver. The fractions, or combination of fractions, were incubated with N-hydroxy-FAA in Medium B, as described in the text. The substrate concentration in all experiments was 0.070 Fmole per ml. I-Hydroxy-FAA and 3-hydroxy-FAA were determined by the spectrophotometric method.

* 3-Methylcholanthrene (2.0 mg/lOO g of body weight) was injected intraperitoneally 24 hours prior to preparation of the cell fractions.


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1738 Isomerixation of N-Hydroxy-2,fEuorenylacetamide to o-Amidophenols in Vitro Vol. 244, Tu’o. 7

TABLE VI

Effect of dialysis of soluble fraction from rat liver on isomerization of N-hydroxy-FAA to I-hydroxy-FAA and S-hydroxy-FAA

The cell fractions from 4.0 g of wet liver of 3-methylcholanthrene-treated rats were incubated in 0.01 M phosphate buffer, pH 7.4, with N-hydroxy-FAA (0.14 rmole per ml of incubation mixture). The quantities of 1-hydroxy-FAA and of 3-hydroxy- FAA formed were determined spectrophotometrically as described in the text.

Cell fraction assayed

600 X g supernatant liquid (undialyzed) Dialyzed soluble fraction + microsomal-

mitochondrial sediment”. Dialyzate + microsomal-mitochondrial sedi-

ment....................................

% 4.35 1.29

6.74 1.95

0.66 <0.30

a The soluble fraction (22.5 ml) was placed into Visking NoJax cellulose casing size 18 and dialyzed in a rocking dialyzer for 16 hours against 1.6 liters of deionized water. The dialyzing liquid was collected and concentrated to 22.5 ml by lyophilization. The concentrate is referred to in the table as the dialyzate.


TABLE VII

Evidence for relation between enzymatic isomerization and reduction of N-hydroxy-FAA by fractionated soluble proteins of rat liver

combined withmicrosomal-mitochondrial fraction from liver of 3-methylcholanthrene-treated rat

Cell fraction testeda, ’

Subfraction A + microsomal- mitochondrial sediment

Subfraction B + microsomal- mitochondrial sediment

Subfraction C + microsomal- mitochondrial sediment

Subfraction D + microsomal- mitochondrial sediment

Subfraction E + microsomal- mitochondrial sediment


m/moles/mg proteinlhr

0.36 0.48 4.81

0.93 2.30 16.19

0.92 1.10 12.15

1.66 3.20 24.48

0.80 0.75 8.48

(FAA + FM/ (I-hydroxy-FAA + 3-pAgmY-

5.7

5.1

6.0

5.2

5.5 (5.5 f 0.3)d

a Subfractions A, B, C, D, and E correspond to the groups of proteins obtained by ion exchange chromatography of the soluble fraction of rat liver on DEAE-cellulose as indicated in the elution profile of Fig. 4.

b The microsomal-mitochondrial sediment was prepared from 4.0 g of wet liver of 3-methylcholanthrene-treated rats as described in the text.

c 1-Hydroxy-FAA, 3-hydroxy-FAA, FAA, and 2-fluorenamine (FA) were isolated from the incubation mixtures of N-hydroxy- FAA-g-W (0.14 rmole per ml of incubation mixture) with the cell fraction as shown in Fig. 2. The amounts of these metabolites were estimated by inverse isotopic dilution.

d The values in parentheses are the mean and the average deviation from the mean.

exhibited the highest specific activities with respect to the formation of the o-hydroxylated and reduced metabolites (Table VII). It was calculated that the ratio of the sum of the reduced metabolites to the sum of the o-hydroxylated metabolites was constant (&lo%) for all fractions (Table VII). This observation sug-

gested that isomerization and reduction were catalyzed by the same enzyme or involved a common intermediate.

DISCUSSION

The induced isomerization of N-hydroxy-FAA by rat liver has been tentatively formulated by the two-step reaction sequence shown in Fig. 8. In the first step, N-hydroxy-FAA is dehydroxylated by an enzyme in the soluble fraction to a positively charged amidonium ion which may subsequently undergo reduction or isomerization. The dehydroxylation was assigned to the soluble fraction rather than to the particulate fraction, because reduction of N-hydroxy-FAA to F,4A, which involves a dehydroxylation (27) and the addition of a hydride ion, was performed by the soluble fraction at a 25-fold faster rate than by the microsomal-mitochondrial sediment from the livers of 3-methylcholanthrene-treated rats (Table VIII). The dehydroxylating enzyme was most active in a protein fraction eluted from DEAE- cellulose at pH 4.6 (Fraction D). The presence of considerably smaller amounts of dehydroxylating activity in other protein fractions may be due to the stepwise elution of the proteins from DEAE-cellulose. Accordingly, additional procedures will be used for the purification of the dehydroxylating activity. The fact that chromatography did not separate the soluble proteins into fractions which catalyzed either reduction or isomerization

alone supports the view that a single compound (i.e. the amidonium ion) was the intermediate common to both reactions. The observation that the products of both reactions were, in all fractions, formed in a constant proportion (Table VII) likewise argues for this interpretation of the data. The role of the induced microsomal enzyme remains at present conjectural. It may complete the isomerization by catalyzing the nucleophilic attack of hydroxyl ions from the medium on the positive carbon atoms 1 and 3 of the fluorene nucleus as shown in Fig. 8. Alter- natively, the induced enzyme may stabilize the amidonium ion and its resonance forms. In this event, the addition of hydroxyl ions to the electrophilic centers at carbon atoms 1 and 3 may proceed nonenzymatically.

The formation in vitro of an amidonium ion derived from N-hydroxy-FAA has been inferred previously (16, 28). In these experiments, the amidonium ion arose from the nonenzymatic decomposition of the acetate ester of N-hydroxy-FAA (16). Sulfate and phosphate esters of N-hydroxy-FAA have also been considered as the immediate precursors of the amidonium ion (29). The present data suggest the direct dehydroxylation of N-hydroxy-FAA, not requiring previous esterification, as an alternate mechanism for the formation of the amidonium ion.

Two lines of evidence indicate that the amidonium ion was not derived from an ester of N-hydroxy-FAA in the present experiments. The first is that the isomerization of N-hydroxy-FAA to I-hydroxy-FAA and 3-hydroxy-FAA proceeded maximally in phosphate buffer, Tris buffer, or deionized water. In contrast, the formation of a phosphate or sulfate ester of N-hydroxy- FAA by preparations of rat liver in vitro has been reported to require high concentrations of ATP and of divalent magnesium and sulfate ions (30). The second line of evidence came from the experiments cited above (Table III) which indicated that


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'y-

OH

OH

FIG. 8. Postulated pathways of the isomerization and reduction of A-hydroxy-FAA by rat liver in vitro

TABLE VIII

Formation of F-4 A jrom iVhydroxy-FAA-9-‘4C by cell fractions or combinations of cell fractions from livers of S-methylcholanthrene-

treated or untreated ruts

The cell fractions from 4.0 g of wet liver were incubated in 0.01 14 phosphate buffer, pH 7.4, with A-hydroxy-FAA+‘% (0.070 pmole per ml of incubation mixture). The quantities of FAA formed were determined by inverse isotopic dilution as described in the text.

I Assay of cell fractions or of combination of cell fractions Conversion

Experi- ment Obtained from the Obtained from the

3.MC-treated rata untreated rat

d$%?&

% 1 600 X g supernatant liquid 4.48 2 iLlicrosomal-mitochondrial Soluble fraction 4.43

sediment 3 Microsomal-mitochondrial 0.71

sediment 4 Soluble fraction 17.12

a 3-Methylcholanthrene (3.MC) (2 mg/lOO g of body weight) was injected intraperitoneally 24 hours prior to the preparation of the cell fractions.

N-hydroxy-FAA was a better sub&rate for the induced iaomeri- zation than was its acetate ester. In view of these findings it seems unlikely that N-acetoxy4fluorenylacetamide or similar esters are obligatory intermediates for the formation of the amidonium ion and for the subsequent isomerization.

Contrary to the report of Booth and Uoyland (7) the evidence obtained here indicates that the isomerization of N-hydroxy- FAA to the o-amidofluorenols is not NADPH-dependent and that it requires the combined action of an enzyme in the soluble fraction and of an induced enzyme in the microsomes. The discrepancies between these findings and those of Booth and Boyland are difficult to reconcile. It is possible that the compound which they isolated from their incubation systems and reported to be l-hydroxy-FAA was in fact not 1-hydroxy-FAA. Booth and Boyland based the identification of the compound solely on its mobility on thin layer chromatograms and on color reactions with spray reagents. It seems to us that these criteria are insufficient to establish the identity of l-hydroxy-FAA unequivocally.

The question may be raised whether the isomerization of N- hydroxy-FAA plays a role in the carcinogenic activity of the nrylhydroxamic acid. It would appear, at first glance, that this question cannot be answered in the affirmative since the reaction requires, at least in liver in vitro, an inducer and since no endoge-

nous compound has as yet been found which would serve this function. Even if such an inducer were found, the products of the isomerization, I-hydroxy-FAA and 3-hydroxy-FAA, when tested for carcinogenic activity, were inactive (15, 31). How- ever, the experiments presented here provide indirect evidence for the formation of an amidonium ion, by the enzymatic dehydroxylation of N-hydroxy-FAA, without involving intermediate acyl esters of N-hydroxy-FAA. The same amidonium ion arising from the spontaneous decomposition of acyl esters of N-hydroxy-FAA has been shown to arylate various tissue nucle- ophiles (28, 32), and the arylamidation of nucleic acids or proteins, or both, by this unstable intermediate is currently presumed to be one of the initial molecular events in the induction of neoplasia by N-hydroxy-FAA (28). The idea that the amidonium ion derived from N-hydroxy-FAA by enzymatic dehydroxylation is the common intermediate for the reduction and isomerization of the arylhydroxamic acid as well as for the arylamidation of tissue constituents will be further pursued.

Acknowledgments-The authors thank Mrs. B. Zakis and Mrs. J. Thaker for technical assistance and Dr. E. J. Barry of this laboratory for help with the ion exchange chromatographies and for discussion.

REFERENCES

1. MILLER, E. C., AND MILLER, J. A., Biochim. Biophys. Acta, 40, 380 (1960).

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H. R. Gutmann and R. R. EricksonREACTION

: AN INDUCIBLE ENZYMATICin Vitro-Amidophenols by Rat Liver o-Hydroxy-2-fluorenylacetamide to NThe Conversion of the Carcinogen

1969, 244:1729-1740.J. Biol. Chem.

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